1. Field of the Invention
The present invention generally relates to inhibitors of anthrax lethal factor protease activity.
2. Description of the Related Art
Anthrax, a disease caused by Bacillus anthracis, has recently been the subject of intense interest because of its use as a biological weapon against human populations.
The inhalation of B. anthracis spores is often fatal if the condition is not properly diagnosed and treated with antibiotics during the early stages of infection. In many cases antibiotic regimes may not be effective, especially if there is bacterium overload, which causes large amounts of lethal toxin to be released. Hence, a new level of adjunct treatment is needed to inactivate the toxins released by B. anthracis. 
Anthrax toxin (AT) consists of three proteins: lethal factor, protective antigen and edema factor, all of which work in concert to kill host cells. Initially, PA binds to an AT receptor on the host cell surface, where it is subsequently cleaved by furin (or furin-like proteases) to produce a 20-kDa N-terminal fragment (PA20) and a 63-kDa C-terminal fragment (PA63). See Bradley, K. A., et al. (2001) Nature 414:225-229; Scobie, H. M., et al. (2003) PNAS USA 100:5170-5174; Klimpel, K. R., et al. (1992) PNAS USA 89:10277-10281; and Molloy, S. S., et al. (1992) J. Biol. Chem. 267:16396-16402. After cleavage, seven PA63 monomers assemble to form a heptameric prepore capable of binding both LF and EF. Upon binding of LF or EF, the entire complex undergoes receptor-mediated endocytosis. It is hypothesized that the acidic endosomal environment causes a conformational change in the PA63 heptamer to produce a functional pore that traverses the membrane and translocates the two enzymatic moieties LF and EF into the cell cytosol. EF is a calmodulin-dependent adenylate cyclase; LF is a Zn-dependent metalloprotease that cleaves several members of the MAPKK family near the N terminus. See Leppla, S. H. PNAS USA (1982) 79:3162-3166; Vitale, G. et al. (1998) Biochem. Biophys. Res. Commun. 248:706-711; and Duesbery, N. S. et al. (1998) Science 280:734-737. This cleavage prevents interaction with, and phosphorylation of, downstream MAPK, thereby inhibiting one or more signaling pathways. See Duesbery, N. S. et al. (2001) PNAS USA 98:4089-4094. Through a mechanism that is not yet well understood, this results in the death of the host. Recent studies suggest that the inactivation of p38 MAPK induces apoptosis in LF-exposed macrophages, thereby preventing the release of chemokines and cytokines, and preventing the immune system from responding to the pathogen. See Park, J. M., et al. (2002) Science 297:2048-2051. Based on the current understanding of the mechanism of anthrax toxin, methods may be developed to inhibit various steps in toxin assembly and/or function. In one antitoxin therapy approach, dominant-negative PA mutants have been generated that coassemble with the wild-type PA protein, blocking the translocation of LF and EF across the cell membrane. Such PA mutants are potent inhibitors of anthrax toxin in both cell-based assays and in vivo animal models. See Sellman, B. R., et al. (2001) Science 292:695-697; and Singh, Y., et al. (2001) J. Biol. Chem. 276:22090-22094. In a second approach, a peptide inhibitor that binds to the heptameric PA and prevents the interaction of PA with LF and EF has shown efficacy in animals. See Mourez, M. et al. (2001) Nat. Biotechnol. 19:958-961.
The lethal action of anthrax toxin may also be inactivated by molecules that inhibit the protease activity of LF. So far, the only known small molecule inhibitors of LF are nonspecific hydroxymates that are effective at greater than about 100 μM concentration and more recently reported hydroxymate derivatives of peptide substrate that inhibit LF at nanomolar concentrations. See Hammond, S. E. & Hanna, P. C. (1998) Infect. Immun. 66:2374-2378; and Tonello, F., et al. (2002) Nature 418:386; Turk, B. E. et al. (2004) Nat. Struct. Mol. Biol. 11(1):60-66. Unfortunately, no small molecule (non-peptidic) inhibitors of anthrax lethal factor, which are effective in the low μM range, have been reported.
Thus, a need exists for more effective compounds and methods for preventing intoxication by anthrax lethal factor.